At present, the study of the epidemiology, treatment and prophylaxis of human, animal and plant diseases is a priority objective of human and veterinary medicine and of agronomy or the plant hygiene industry. Identification and quantification of microorganisms in media such as tissues, biological fluids and in-vitro preparations are steps that are indispensable for the proper conduct of such projects. Taking into account the size of most microorganisms, particularly viruses, and their concentration in biological media (generally of the order of 10.sup.1 to 10.sup.12 microorganisms per mL), the only current technological tools that permit both molecular identification and quantitative evaluation are those which make use of genome fragment amplification. The method of genomic amplification most frequently used at the present time is called polymerase chain reaction or PCR. Thus, it is possible, by means of an enzyme, polymerase, to amplify a DNA-containing genome fragment. Likewise, it is possible to carry out a reverse transcription of RNA into DNA, and an amplification with the aid of the reverse transcriptase+polymerase couple or by an enzyme performing these two operations. Once amplified, this genome fragment or "amplificate" is specifically identified by various methods (radioactive or colored probe, fragment size).
Methods of quantitation of HIV by PCR are described, e.g., by M. Holodniy et al., J. Infect. Dis. 163, 862-866 (1991); S. Aoki-Sei et al., J. AIDS Res. Hum. Retrovirus 8, 1263-1270 (1992); P. Bagnarelli et al., J. Virol. 66, 7328-7335 (1992); M. Piatak Jr. et al., Science 259, 1749-1755 (1993); S. M. Bruisten et al., AIDS 7 (Suppl. 2), S15-S20 (1993).
Methods of quantitation of hepatitis C virus RNA have been described, e.g., by U. Kumar et al., J. Virol. Methods 47(1-2), 95-102 (1994); A. Mazin et al., J. Clin. Microbiol. 32(8), 1939-44 (1994); A. Ravaggi et al., J. Clin Microbiol. 33(2), 265-9 (1995); K. K. Young et al., J. Clin Microbiol. 33(3), 654-7 (1995).
Methods of quantitation of hepatitis B virus DNA have been described, e.g., by P. Lehtovaara et al., PCR Methods Appl. 3(3), 169-75 (1993); J. Wu et al., J. Virol. Methods 49(3), 331-41 (1994); H. L. Zaaijer et al., J. Clin Microbiol. 32(9), 2088-91 (1994); S. Kaneko et al., J. Clin. Microbiol. 27(9), 1930-33 (1989).
However, the relationship between amplified product and initial DNA or RNA is not really constant from one experiment to another; hence, the necessity of being concerned with the problem of measuring a standard whose concentrations, moreover, are known.
Until now, the PCR amplification capacities could be evaluated by means of cells wherein the genome to be determined is present in a constant number of copies only, or by means of plasmids each having a single gene fragment. In this way, it could be established that, thanks to PCR, even a single gene fragment can be specifically identified by PCR. It has also been established that a proportionality ratio exists between DNA concentrations of a plasmid that are subjected to amplification and the DNA concentrations measured after amplification. However, this proportionality ratio has been found to change from one experiment to another, because PCR amplification is a biological process, resulting from the action of polymerase, which is not controlled in such complete fashion as to make it purely physical technique.
This has led researchers and engineers of biotechnological companies to introduce standardization into every viral quantitation experiment. Until now, the standard has consisted in a series of concentrations (generally three to five) of a standard plasmid whose DNA fragment to be amplified has the same primers, and, depending on the technologies, is either of a partly different sequence or of identical sequence. The amplification ratio of the DNA of equal concentrations of two plasmids of identical primers but of different lengths and thus of different sequences (1) may be different from one case to another and (2) is not necessarily identical to that of the DNA originating from the identical native virus concentrations within the same experiment.
The problem is even more complex if it is sought to quantitate or identify RNA viruses, such as, e.g., that of AIDS (HIV). For RNA viruses, the PCR amplification step must, in effect, be combined with a reverse transcription step (abbreviated RT) which is frequently carried out either with the aid of another enzyme, reverse transcriptase, or with an enzyme possessing both actions (reverse transcription and DNA amplification). In both cases, the enzymes currently available on the market make it possible to obtain reverse transcription ratios that can vary from one experiment to another from 10:1 (that is to say, 10 copies of RNA give one copy of DNA) to 100:1 (100 copies of RNA give 1 copy of DNA). Until now, the resulting problem, namely the noncomparability of results obtained in successive experiments, has been solved by simultaneous incubation of a concentration range of a standard RNA fragment (standard transcript) using the same primers, but whose amplified part is either identical if it is used as an external standard, or different in sequence or length if it is used in co-amplification, the transcript then being incubated in the same tube as the RNA of the target microorganism to be determined. Unfortunately, it seems that the concentrations of two transcripts can be different, and be different even from those originating from RNA obtained from the identical virus concentrations.
In conclusion:
The known reverse transcription and/or amplification techniques do not furnish identical results from one experiment to another for the same sample; PA1 It is imperative to use a standard, which may be either internal or external; PA1 An external standard composed either of a plasmid for a DNA microorganism or a transcript RNA for an RNA microorganism permits only a relative quantitation, because the amplification ratio is not necessarily identical to that of the DNA or RNA of the complete microorganism; PA1 Moreover, a co-amplified internal standard involves different lengths or sequences of the plasmid or the transcript, and has the same drawbacks as an external standard using a plasmid or a transcript. PA1 (1) one knows or is otherwise able to determine a standard concentration of the microorganism or the concentration of the DNA or RNA contained in this microorganism, called standard microorganism; and PA1 (2) one compares the quantity of the product of reverse transcription and/or amplification of the RNA or DNA originating from an unknown concentration of the microorganism with amplificate quantities originating from the RNA or DNA of several known concentrations of said microorganism. PA1 1) A certain quantity of the microorganism to be determined is taken from human, animal or plant samples or from cell cultures, and then purified, the quantity of microorganism being advantageously sufficient to ensure that the target DNA or RNA concentrations are measurable by direct methods; PA1 2) The DNA or RNA is extracted and the quantity collected (expressed in ng or .mu.g) is measured; PA1 3) In other respects, the number of nucleotide bases of the microorganism in question is made use of, this number being known, e.g., from databanks, or be determinable by conventional methods; PA1 4) The total molecular mass of the DNA or RNA of said microorganism is calculated from this number of nucleotide bases, keeping in mind the mean molecular mass of one nucleotide, which is also known (about 330 daltons); PA1 5) Using Avogadro's number (6.02.times.10.sup.23), the quantity of DNA or RNA of each microorganism is calculated in this manner; PA1 6) A range of standard microorganism concentrations obtained by successive dilutions of the microorganism previously standardized according to its DNA or RNA concentrations is prepared and/or used, to form an external standard for which (a) the DNA or RNA concentration and (b) the microorganism concentration is thus known for each dilution; PA1 7) The DNA or RNA of the standard and target microorganisms is extracted, and the extracts are subjected concurrently to a revealing by means of the so-called branched DNA method or any other revealing method which does not amplify the DNAs or the RNAs of the standard or of the target microorganism and/or to reverse transcription and/or to at least one amplification and the resulting values are recorded; and PA1 8) The concentrations of DNAs or RNAs of both the standard and the target microorganisms are compared or the amplification products of the target microorganisms are compared with those of the external standard, to deduce the DNA or RNA or total microorganism concentration values in each target microorganism sample. PA1 of the same inactivated microorganism whose DNA or RNA remains capable of detection or quantitation by means of a revealing method such as the so-called branched DNA revealing method and/or reverse transcription and/or amplification in a manner identical to the DNA or RNA of native virus; PA1 of a complete nucleotide sequence, extracted from the same microorganism; or PA1 of a complete nucleotide sequence produced by synthesis. PA1 A series of known standardized concentrations of the complete DNA or RNA microorganism to be quantitated or detected, or of DNA or RNA concentrations corresponding to known concentrations of said microorganism; PA1 Means of extraction, revealing and/or reverse transcription and/or amplification, notably by a PCR, RT-PCR or NASBA amplification of the DNA or RNA of the target and standard microorganism; PA1 Means for analysis of the DNA or RNA of the target and standard microorganism or of the revealing or amplification products.
In the present state of the art, amplification of the DNA of a plasmid is not truly proportional to that undergone by viral DNA. As for the standard currently used for RNA viruses, it is a transcript which has the same drawbacks as a plasmid, to which is added the possibility of free RNA degradation.
Hence, there has been a need for microorganism quantitation and detection tests yielding absolute values of the number of target microorganisms.